Lignin and two polysaccharides hemicellulose and cellulose form the three major components of plants physiology and are collectively called as lignocellulose. Of these three, cellulose and hemicellulose are basically polymers of sugar monomers like glucose, xylose, galactose, arabinose etc. Therefore, cellulose and hemicellulose derived from plant residues, if hydrolyzed to monomeric sugars, can form a useful and abundant renewable source of raw material for a variety of useful chemicals and biochemicals. Conversion of this generally tightly compacted composite lignocellulosic material to sugar is accomplished by a composite process known as hydrolysis and saccharification. Worldwide research on saccharification processes for the conversion of lignocelluloses to sugars has followed three major approaches. First is chemical hydrolysis, the second is thermal hydrolysis and the third is enzymatic hydrolysis.
In a general chemical hydrolysis process, hemicellulose is separated in the first step from the lignocellulose composite material by the action of an acid or alkali. The plant material/mass is mixed with a dilute solution of an acid or alkali and then heated. This process releases and “hydrolyzes” the hemicellulose. Hydrolysis of hemicellulose produces pentose sugars (C5 sugars) as well as some hexose sugars (C6 sugars). The second step is a higher temperature acid hydrolysis process that hydrolyzes the plant material cellulose, producing almost solely C6 (hexose) sugars, and lignin. The C6 sugars, when separated substantially from lignin, are readily fermentable, and the recovered lignin can be used for process heat or making other products.
Two stage acid hydrolysis processes have been used for many years. However, it is now known that the acid processes also produce chemicals other than sugars that not only represent a process loss but also lead to problems later in the use of the sugars in downstream processes like fermentation to useful products like lactic acid, alcohols, organic acids etc. Another major problem with these systems has been that the acid must either be recovered for re-use or it must be neutralized through the use of lime in order to mitigate effluent and pollution problems.
Autothermal processes on the other hand do not make use of any chemicals and thus are cleaner processes. High temperatures and short exposures like used in Steam Explosion processes, results in breakdown of the lignocellulosic biomass into monosugars and hydrolyzed lignin. However, such processes suffer from the drawbacks of lower sugar yields, formation of unwanted side-products that are inhibitory to downstream processes, and are energy intensive.
Use of enzymes, generally preceded by some or the other mild pretreatment steps, provides much cleaner and low energy process for cellulose and hemicellulose hydrolysis and saccharification and finally provides better quality end products i.e. sugars in higher yields.
Several enzymes are known to specifically, or non-specifically, hydrolyze plant cell wall polysaccharides. Such enzymes derived from culture filtrates of microorganisms have found large scale applications for hydrolysis of cell wall components (Reese, E. T. et al, Can. J. Microbiol. 19, 1973, 1065-1074). Microorganisms produce numerous proteins, and some also produce cellulose and/or hemicellulose splitting enzymes. Most reports and technologies make use of these catalytic enzymes in free soluble form that cannot be recovered for reuse. Further, often the substrates namely cellulosic and/or hemicellulosic polymers and products of hydrolysis thereof, have tendencies to ‘inhibit, the enzymes’ actions. Such a use of these enzymes makes them less attractive for use on a commercial scale or makes the use of the enzymes more expensive than often desired. Therefore, for reasons of cost, the amount of enzymes used per unit weight of cellulose and/or hemicellulose hydrolyzed is often kept to a minimum, which in turn reduces the rate of hydrolysis reactions and increases the reaction times.
For the reasons stated above, and for other reasons stated below which will become apparent to those skilled in the art upon reading and understanding the present specification, there is a significant need in the art for systems and methods that provide for improved biomass conversion to sugars in a cost-effective manner. Shortcomings of the enzyme process can be alleviated making it the obvious choice for new process development.
Cellulose and hemicellulose are the first and second most abundant polysaccharides in nature. Cellulose represents anywhere from 30 to 60% while hemicelluloses represent about 20-35% of lignocellulosic biomass (LBM) such as corn fiber, corn stover, wheat straw, rice straw, and sugarcane bagasse. While cellulose is an almost homogeneous polymer comprised of several hundreds to thousands D-glucose units linked through 1,4β-glycosidic linkages, hemicelluloses are heterogeneous polymers of pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose), and sugar acids. Hardwood hemicelluloses contain mostly xylans, whereas softwood hemicelluloses contain mostly glucomannans. Xylans of most plant materials are thus heteropolysaccharides with homopolymeric backbone chains of 1,4-linked β-D-xylopyranose units. Besides xylose, xylans may also contain arabinose; glucuronic acid or its 4-O-methyl ether; and acetic, ferulic, and p-coumaric acids. The frequency and composition of branches are dependent on the source of xylan while the backbone consists of O-acetyl, α-arabinofuranosyl, α-1,2-linked glucuronic or 4-O-methylglucuronic acid substituents.
For both cellulose and hemicellulose components to be efficiently converted to their monosugar components these must first be extracted from the lignocellulosic complex. Enzymatic saccharification of these two components using cellulases and hemicellulases is the preferred method due to rapid action of the enzyme, and negligible substrate loss and side product generation. Both cellulose and hemicellulose in intact LBM however, are not accessible to enzymatic hydrolysis. And therefore pretreatment of the LBM to render these amenable to enzyme action is mandatory (Himmel, M. E. et al, 2007; Bothast and Saha, 1997). While cellulose, though a homopolymer, is a far more bulkier, crystalline and compact molecule, the structure of hemicellulose is more complex as it comprises of pentoses, some hexoses and side chain groups such as acetyl and uronic acids. Thus, enzymatic hydrolytic action for both cellulose and hemicellulose requires combined action of more than one enzyme. For cellulose hydrolysis the crystal structure of cellulose needs to be partially or wholly rendered amorphous after which a mixture of exo and endo cellulases is required for conversion of the polymeric cellulose to much smaller oligomeric molecules. On the other hand, in case of hemicellulose, the presence of side chain groups hampers the action of major backbone depolymerizing enzymes i.e. exo and endo xylanases, and mannanases. To address this problem accessory enzymes such as α-L-arabinofuranosidase, α-glucuronidase, acetylxylan esterase, ferulic acid esterase, and p-coumaric acid esterase which have the ability to hydrolyze the side chains have to be present with the major hemicellulases to achieve complete degradation of hemicellulose to obtain high yields of monosaccharide sugars (Biely and Tenkanen, 1998).
As a result of such scenario, cellulase and hemicellulase preparations used for depolymerizing or hydrolyzing cellulose and hemicellulose, respectively, contain a myriad of major and minor enzymes that all act together.
However, on the other hand, it is now well recognized that, the starting and intermediate substrates occurring during the sequential but complicated process of polymer hydrolysis, tend to act as partial or complete inhibitors of the enzymes present in the mixture preparations used (Beguin P et. al, (1994), FEMS Microbiological Review, 13, 25-58 and Ven H Tilbeurgh et al, Studies of the cellulytic system of Trichoderma reesei QM 94014 (1989), European journal of Biochemistry, 189, 553-559). As a result of this fact, and the fact that one may not want to use excessive quantities of enzymes for cost reasons, the enzymatic saccharification processes for both cellulose and hemicellulose are long duration reactions requiring 24 to 48, and often more, hours for completion. It has long been accepted that enzymes are truly efficient catalysts. However, since derived from biological sources and purified, at least partially, and on account of their inherently complex, fragile and sensitive nature, enzymes are expensive and unstable. This has put severe limitations on the spectrum and scale of applications of enzymes in industry (F. Dourado et al, 2002, Journal of Biotechnology, 99, 121-131). Several methods have been devised to render the enzymes stable and less expensive for use for production scale applications. Thus, new enzymes, including cellulases and hemicellulases, have been developed and manufactured such that they are stable to wide temperature, pH and other harsh conditions like presence of inhibitors (Khare and Gupta, 1988, Applied Biochemistry and Biotechnology, 16, 1-15, Busto et al, Bioresource Technology, 1997, 60, 27-33). However, despite these efforts, these enzymes today contribute significantly to the cost of conversion of cellulose and hemicellulose to simple sugars.
One way of reducing enzyme cost is to use the enzymes in immobilized form, or in a form, or way, that permits reuse of enzymes over many cycles, or over extended periods of time. Thus, in a reusable form or way, the enzymes are retained in the reactor, while the substrate/s and product/s flow in and out, in batch or continuous fashion. However, use of an enzyme in immobilized form on a solid support, requires that reactants (or substrates) and products are in soluble form to facilitate the reaction. Further, when using enzymes for reactions involving polymeric reactants and products (like cellulose and hemicellulose), the accessibility of the enzymes in the pores of the immobilization support becomes rate limiting and the reactions become too slow to be of practical use (Woodward J. 1989, Journal of biotechnology, 11, 299-311). This, and the fact that cellulose is an insoluble solid and hemicelluloses are polymeric with low solubility in water as well, has prevented use of cellulases and hemicellulases in recyclable and/or immobilized forms.
U.S. Pat. No. 4,200,692 discloses a process for the production of xylose by enzymatic hydrolysis of xylan wherein the enzymes are immobilised separately but incubated together and, the xylan solution is broken to xylobiose and xylose and acid sugars. After 4 hours total hydrolysis to xylose and 4-O-methylglucuronic acid is claimed. US2008/065433 discloses a process for obtaining fuel ethanol by using agricultural and agroindustrial waste materials composed of lignocellulose, and especially sugar cane bagasse. The hemicellulose fraction is submitted to mild hydrolysis with sulphuric acid, and the solid material from this hydrolysis is submitted to a process of saccharification (enzymatic hydrolysis) with simultaneous rapid alcoholic fermentation under conditions which allow a significant increase in conversion to alcohol in a greatly shortened time, approximately 8-32 hrs.
U.S. Pat. No. 6,423,145 discloses a modified dilute acid method of hydrolyzing the cellulose and hemicellulose in lignocellulosic material under conditions to obtain higher overall fermentable sugar yields, comprising: impregnating a lignocellulosic feedstock with a mixture of an amount of aqueous solution of a dilute acid catalyst and a metal salt catalyst, loading the impregnated lignocellulosic feedstock into a reactor and heating for a sufficient period of time to hydrolyze substantially all of the hemicellulose and greater than 45% of the cellulose to water soluble sugars; and recovering the water soluble sugars.
US2009/098618 discloses a method for treating plant materials to release fermentable sugars. Lignocellulosic materials are subjected to disc refining together with enzymatic hydrolysis to produce sugar rich process stream that may subsequently be subjected to fermentation to produce biofuels and chemicals.
U.S. Pat. No. 5,348,871 discloses a process for converting cellulosic materials, such as waste paper, into fuels and chemicals utilizing enzymatic hydrolysis of the major constituent of paper, cellulose. Waste paper slurry is contacted by cellulase in an agitated hydrolyzer. The glucose produced from hydrolyzer is fermented to ethanol in a continuous, columnar, fluidized-bed bioreactor utilizing immobilized microorganisms. The process disclosed in the patent requires ‘many hours to days for acceptable yields’.
U.S. Pat. No. 5,637,502 discloses a batch process for converting cellulosic materials into fuels and chemicals, such as sugars and ethanol, utilizing enzymatic hydrolysis of cellulose. Waste paper slurry is contacted by cellulase in an agitated hydrolyzer. An attritor and a cellobiase reactor are coupled to the agitated hydrolyzer to improve reaction efficiency. Additionally, microfiltration, ultrafiltration and reverse osmosis steps are included to further increase reaction efficiency and recycling of the enzymes. The resulting sugars are converted to a dilute ethanol product in a fluidized-bed bioreactor utilizing a biocatalyst, such as microorganisms. The time of hydrolysis of paper cellulose is about 24 hours.
U.S. Pat. No. 227,162 discloses a method for lignocellulose conversion to sugar with improvements in yield and rate of sugar production by using ionic liquid pretreatment. However, the time required for complete batch enzymatic hydrolysis is within 16 to 36 hours for two of the representative biomass samples—corn stover, poplar which is a substantially longer period.
U.S. Pat. No. 5,932,452 discloses a process for the hydrolysis of a hemicellulose substrate containing xylo-oligomers, obtained from steam exploded plant biomass or enzymatically partially pre-hydrolyzed xylan, with an immobilized enzyme. This process however, has the pre-requisite of producing partially hydrolyzed hemicellulose which in turn needs to be obtained from plant biomass through suitable process such as steam explosion. Steam explosion is a hydrothermal process and is known to produce furfural derivatives that are known to affect both enzymatic conversion, and later fermentation efficiencies.
US2008/076159 discloses methods to produce enzymes or novel combinations of enzymes, which provide a synergistic release of sugars from pre-treated plant biomass. However, the disclosed process does not reduce the saccharification period which is in the range of 24-72 hours.
EP2017349 discloses a method for the direct enzymatic treatment of raw polymeric feedstock and separation of the resulting soluble components. However, there is no mention of recovery and reuse of the enzymes, and the hydrolysis duration is also a prolonged one.
WO/2006/063467 discloses a continuous process system for enzymatic hydrolysis of pre-treated cellulose which comprises introducing aqueous slurry of the pre-treated cellulosic feedstock at the bottom of a vertical column hydrolysis reactor. Axial dispersion in the reactor is limited by avoiding mixing and maintaining an average slurry flow velocity of about 0.1 to about 20 feet per hour, such that the undissolved solids flow upward at a rate slower than that of the liquid. Cellulase enzymes are added to the aqueous slurry before or during the step of introducing. An aqueous stream comprising hydrolysis products and unhydrolyzed solids is removed from the hydrolysis reactor and after solid separation the unhydrolyzed cellulose is recycled. Also provided are enzyme compositions which comprise cellulase enzymes and flocculents for use in the process. In addition, a kit comprising cellulase enzymes and flocculent is described that is said to provide exposure of the enzyme to the substrate. Although the cellulose conversion is better in this case than batch reactor, the time required is 48 hours to 200 at respective enzyme loading of 32 units/g cellulose to 5 units/g cellulose.
WO/2009/004950 discloses that monosaccharide and/or a water-soluble polysaccharide can be produced with a high degree of efficiency by hydrolyzing a cellulose-containing material with a sulfonate-containing carbonaceous material. The used sulfonate-containing carbonaceous material can be reactivated and reused by carbonization and sulfonation, without the need of separating the sulfonate-containing carbonaceous material from the unreacted portion of the cellulose-containing material. This method, which does not use any enzymes, enables to reduce the cost for hydrolysis, can reduce the amount of waste materials, and therefore can contribute to the global environmental conservation.
The concept of enzymatic hydrolysis of cellulose and hemicelluloses is known since long. As described above, most enzymatic hydrolysis processes in use, or reported are batch processes and take 12-48 hrs for complete saccharification. More often, the enzymatic processes remain incomplete resulting in high enzyme cost and slow reactions leading to low throughputs and hence high capital investment in large reactors. While use of higher dosage of enzymes can increase the hydrolysis rate, the cost considerations limit the dosages. Further, dosages of enzymes in typical batch processes are higher than desired on account of inhibitory effects of reaction substrates and products on the enzymes. For this reason, new efficient methods are needed for cellulose and hemicellulose saccharification which will require lower enzyme dosages per kilo of cellulose and hemicellulose, not require high temperatures and pressures, will not generate hazardous byproducts, will be less time consuming, and require less energy, thus making the process more economically viable.
At the scale at which a biomass to sugars plant is expected to operate (typically 100 to 1000 tons biomass/day) large reaction times imply humongous sized enzyme reactors exceeding several 100 KL capacities. It is therefore necessary to speed up the reaction rates thereby increasing volumetric throughputs.